衍生化GC-MS法测定流感病毒裂解疫苗中游离甲醛含量
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摘要
目的:建立测定流感病毒裂解疫苗中游离的痕量甲醛的衍生化GC-MS方法。方法:优化衍生化条件后,利用2,4-二硝基苯肼(2,4-dinitrophenylhydrazine,DNPH)衍生流感病毒裂解疫苗中的游离甲醛,用环己烷萃取衍生物,以GC-MS法测定。气相色谱-质谱条件为:以Agilent DB-5MS毛细管色谱柱分离,初始温度为60℃,以10℃·min-1的速率升温至150℃,保持2 min,再以10℃·min~(-1)升至260℃,保持5 min。检测器为质谱检测器,电子轰击离子化模式,电子能量70 e V,离子源温度280℃,进样口温度为250℃;扫描方式为选择性离子检测,检测离子m/z 79、210;载气为氦气,流速:1. 0 ml·min-1,分流比为2∶1;进样量:1μl。利用建立的方法测定了两个厂家共10批样品中游离的甲醛含量。结果:甲醛浓度在0. 10～30. 00μg·ml~(-1)内与峰面积呈良好的线性关系(r=0. 999 8),该方法的最低检测限为0. 01μg·ml~(-1),定量限为0. 03μg·ml~(-1)。低、中、高3个浓度的平均加样回收率为95. 3%(RSD=3. 4%,n=9); 10批流感病毒裂解疫苗中痕量甲醛含量均低于中国药典2015年版三部中规定的50μg·ml~(-1)。结论:该方法操作简单,检出限低,专属性强,可用于流感病毒裂解疫苗中游离甲醛含量的质量控制。
Objective: To determine the free formaldehyde content in influenza virus split vaccine by derivative GC-MS. Methods:Free formaldehyde in influenza virus split vaccine was derived by 2,4-dinitrophenylhydrazine( DNPH) after the optimization of derivatization conditions. The derivatives were extracted by cyclohexane and determined by GC-MS. An Agilent DB-5 MS capillary chromatographic column was used for the separation at initial temperature of 60℃ rose to 150℃ at a rate of 10℃ ·min~(-1) and maintained for 2 min followed by rising to 260℃ at the same rate and maintaining for 5 min. A mass spectrometry detector was used in a selected ion monitoring mode,and m/z 79 and 210 were selected as the monitored ions. The ionization mode was electron impact,and the electron energy of ionization was 70 eV. The temperature of ion source and injection port was 280℃ and 250℃,respectively. Helium was used as the carrier gas and the split flow was 2 ∶ 1,and the sample loading was 1 μl. The free formaldehyde contents in ten batches of vaccine manufactured by two different manufacturers were determined by using the established method. Results: The concentration of formaldehyde showed a good linear relationship with the peak area within the range of 0. 1-30. 00 μg·ml~(-1)(r = 0. 999 8). The lower detection limit of the method was 0. 01 μg·ml~(-1) and the quantitative limit was 0. 03 μg·ml~(-1). The average recovery of low,medium and high concentrations was 95. 3% with the RSD of 3. 4%( n = 9). All the contents of free formaldehyde in the ten batches of influenza virus split vaccine were less than the standard of 50 μg·ml~(-1) described in Chinese Pharmacopoeia( VolumeⅢ,2015 edition).Conclusion: The developed GC-MS method is simple,precise,stable,accurate and specific,which might be used for the determination of the free formaldehyde content in influenza virus split vaccine.
Objective: To determine the free formaldehyde content in influenza virus split vaccine by derivative GC-MS. Methods:Free formaldehyde in influenza virus split vaccine was derived by 2,4-dinitrophenylhydrazine( DNPH) after the optimization of derivatization conditions. The derivatives were extracted by cyclohexane and determined by GC-MS. An Agilent DB-5 MS capillary chromatographic column was used for the separation at initial temperature of 60℃ rose to 150℃ at a rate of 10℃ ·min~(-1) and maintained for 2 min followed by rising to 260℃ at the same rate and maintaining for 5 min. A mass spectrometry detector was used in a selected ion monitoring mode,and m/z 79 and 210 were selected as the monitored ions. The ionization mode was electron impact,and the electron energy of ionization was 70 eV. The temperature of ion source and injection port was 280℃ and 250℃,respectively. Helium was used as the carrier gas and the split flow was 2 ∶ 1,and the sample loading was 1 μl. The free formaldehyde contents in ten batches of vaccine manufactured by two different manufacturers were determined by using the established method. Results: The concentration of formaldehyde showed a good linear relationship with the peak area within the range of 0. 1-30. 00 μg·ml~(-1)(r = 0. 999 8). The lower detection limit of the method was 0. 01 μg·ml~(-1) and the quantitative limit was 0. 03 μg·ml~(-1). The average recovery of low,medium and high concentrations was 95. 3% with the RSD of 3. 4%( n = 9). All the contents of free formaldehyde in the ten batches of influenza virus split vaccine were less than the standard of 50 μg·ml~(-1) described in Chinese Pharmacopoeia( VolumeⅢ,2015 edition).Conclusion: The developed GC-MS method is simple,precise,stable,accurate and specific,which might be used for the determination of the free formaldehyde content in influenza virus split vaccine.
引文
1 Chen CW,Lee YP,Wang YF,et al.Formaldehyde-inactivated human enterovirus 71 vaccine is compatible for co-immunization with a commercial pentavalent vaccine[J].Vaccine,2011,29(15):2772-2776
2赵铠,章以浩,李河民.医学生物制品学[M].北京:人民卫生出版社,2007:805-816
3中国药典[S].2015年版.三部.附录162-164
4纪宏,马迅,祁进,等.衍生化GC法测定重组乙型肝炎疫苗中游离甲醛的含量[J].沈阳药科大学学报,2015,32(4):289-293
5纪宏,马迅,祁进,等.衍生毛细管气相色谱法测定流感病毒裂解疫苗中游离甲醛含量[J].中国生物制品学杂志,2014,27(8):1097-1102
6张璐,马秋冉,戴青,等.衍生气相色谱法测定鸡疫苗中游离甲醛的含量[J].中国兽药杂志,2016,50(7):30-33
7王潇,谷丽娟,李井涛,等.流感病毒裂解疫苗中游离的痕量甲醛反向HPLC检测方法的建立[J].中国生物制品学杂志,2016,29(11):1210-1214
8涂晶,何亮,方舸,等.吸附无细胞百白破联合疫苗中游离甲醛含量柱前衍生反相高效液相色谱检测方法的建立及验证[J].中国生物制品学杂志,2016,29(12):1337-1340
2赵铠,章以浩,李河民.医学生物制品学[M].北京:人民卫生出版社,2007:805-816
3中国药典[S].2015年版.三部.附录162-164
4纪宏,马迅,祁进,等.衍生化GC法测定重组乙型肝炎疫苗中游离甲醛的含量[J].沈阳药科大学学报,2015,32(4):289-293
5纪宏,马迅,祁进,等.衍生毛细管气相色谱法测定流感病毒裂解疫苗中游离甲醛含量[J].中国生物制品学杂志,2014,27(8):1097-1102
6张璐,马秋冉,戴青,等.衍生气相色谱法测定鸡疫苗中游离甲醛的含量[J].中国兽药杂志,2016,50(7):30-33
7王潇,谷丽娟,李井涛,等.流感病毒裂解疫苗中游离的痕量甲醛反向HPLC检测方法的建立[J].中国生物制品学杂志,2016,29(11):1210-1214
8涂晶,何亮,方舸,等.吸附无细胞百白破联合疫苗中游离甲醛含量柱前衍生反相高效液相色谱检测方法的建立及验证[J].中国生物制品学杂志,2016,29(12):1337-1340